The invention concerns a bioreaction module for biochemical reactions, particularly for cell-free biosyntheses of proteins or other polypeptides. For biotechnological applications, biochemical reactions are often carried out in small, easily handled reaction vessels, designated here as bioreaction modules. They primarily serve for the biosynthesis of polypeptides such as peptide hormones, antibiotics and most recently of particular importance, human proteins. Biochemical systems used in such processes include pure translation systems with which the biosynthesis is based on the genetic code of a messenger RNA (mRNA), and transcription/translation systems which also include the preceding step of forming messenger RNA from DNA. In addition to biosynthesis, bioreaction modules are also used for other biochemical reactions, particularly for enzyme reactions.
Cell free biosynthesis is particularly important and has substantial advantages compared to in vivo-systems. For example, it facilitates the expression of toxic and unstable gene products. Cell free synthesis is often used for analytic applications, particularly for rapid and straightforward verification that a desired gene product has actually been synthesized from a cloned gene. There are a plurality of other important applications such as the investigation of protein:protein interactions and protein:DNA interactions.
A method for cell-free biosynthesis of polypeptides is described e.g. in U.S. Pat. No. 5,478,730. The translation system used therein contains a source of MRNA which encodes the polypeptide. The cell-free synthesis system also contains ribosomes, tRNA, amino acids, ATP and GTP. Translation of the mRNA with the assistance of the tRNA leads to the production of the polypeptide, wherein byproducts and waste products of low molecular weight also simultaneously occur. These can pass into the supply chamber through a semi-permeable membrane which separates the chamber containing the synthesis system from this supply chamber. The supply chamber holds a liquid acting as a supply agent and containing ATP, GTP and amino acids. These components are introduced into the synthesis system through the semi-permeable membrane to replace consumption during the bioreaction. Passage of these components through the semi-permeable membrane is possible, because their molecular weight is less than the molecular cutoff. At the same time, biochemical reaction products and other substances whose molecular weight is below the molecular cutoff of the barrier pass out of the reaction chamber and into the supply chamber. The semi-permeable membrane is, in accordance with U.S. Pat. No. 5,478,730, an ultrafiltration membrane embodied as hollow membrane fibers.
U.S. Pat. No. 5,478,730 contains detailed additional description of suitable compositions for the synthesis system and for the supply liquid. Towards this end, the present invention refers to the previous prior art as, in particular, disclosed in this US patent as well as to the literature cited therein. The complete disclosure of these documents is hereby incorporated by reference.
The components which are biochemically active in biochemical reactions can be designated as the xe2x80x9cproducing systemxe2x80x9d. To the extent that the invention is utilized for biochemical reactions other than polypeptide syntheses, a producing system is utilized which is appropriate for the associated application. Further details are given in the extensive literature of this art. In principle, a producing system can be any system which is suitable to facilitate or accelerate the production or synthesis of desired compounds using biochemical means. In addition to the translation and transcription systems mentioned, other possibilities include enzyme and enzyme complexes in combination with the conventional auxiliary substances used in methods of this type.
Since the mRNA required for the biosynthesis of polypeptides is usually only available in very small amounts, the bioreaction modules are often very small. The invention is directed, in particular, to micro bioreaction modules with a chamber containing the producing system (xe2x80x9csystem chamberxe2x80x9d) having a volume preferentially not in excess of 2 ml but in any event less than 10 ml. Since micro bioreaction modules of this type are disposable products intended for one-time use only, it is particularly important that they be manufactured at low cost.
A particular problem when carrying out biochemical reactions in bioreaction modules is to achieve as high a yield as possible, i.e. as large an amount of product as possible relative to the amount of mRNA used. In order to achieve this goal, U.S. Pat. No. 5,478,730 describes a method known to those of skill in the art as CFCF (continuous flow cell-free) synthesis. In this method, the producing system is enclosed in an ultrafiltration cell and the supply liquid is pumped through the semi-permeable membrane into the chamber in which the producing system is located. In such methods the yield is substantially dependent on the amount of supply liquid fed to the producing system (feed rate) per unit time. For example, U.S. Pat. No. 5,478,730 describes an experiment with a synthesis chamber volume of 1 ml using a feed rate varying between 2 ml/h and 3 ml/h. The results show that the higher feed rate results in a high production efficiency, whereas with the lower feed rate the amount of product increased only negligibly.
The CFCF system facilitates high product yields (in excess of 100 xcexcg for a system chamber volume of 1 ml). However, there are substantial associated disadvantages. For example, J. Davies et al.: xe2x80x9cIn vitro Transcription/Translationxe2x80x9d; Promega Notes 56, 14-21, criticizes the CFCF method for various problems caused by blockage of the membrane, bothersome protein aggregation and unexplained interruption of the translation. In addition, a complicated apparatus is required. For these reasons, the authors of this publication propose use of the so-called xe2x80x9cDispoDialyser(copyright)xe2x80x9d. This is a small tubular vessel having semi-permeable walls in which 250 xcexcl of the producing system is filled. In order to carry out the biosynthesis, the DispoDialyser is placed in a conical 15 ml test tube which contains between 3.5 and 7 ml of supply liquid. The test tube containing the DispoDialyser is agitated during the reaction time (20 hours) using a laboratory shaking device.
This procedure attempts to avoid the disadvantages of the CFCF method and is intended to facilitate high production yields using relatively simple apparatus. However, these goals are achieved to only a limited extent. In addition, handling is difficult and the quality of the synthesis result is quite irregular. In many cases, leaks or other mechanical damage to the membrane occur which lead to unusable results.
It is therefore an object of the invention to create a bioreaction module with which bioreactions can be carried out in a simple, economical manner to produce as high a yield as possible.
This purpose is achieved with a bioreaction module for biochemical reactions, in particular for cell-free polypeptide biosynthesis, having a housing comprising a system chamber and a supply chamber, wherein the system chamber contains a producing system during the biochemical reaction and the supply chamber contains a supply liquid during the biochemical reaction and the system chamber and the supply chamber are separated by a semi-permeable membrane, characterized in that the housing comprises a system chamber element and at least one supply chamber element and the semi-permeable membrane is mounted between the chamber elements, such that the system chamber is defined by the system chamber element and the semi-permeable membrane and the supply chamber is defined by the supply chamber element and the semi-permeable membrane. The invention is also directed to a bioreaction method using a module of this type.
A bioreaction module in accordance with the invention comprises only a few parts which are easily produced and assembled. Despite the associated low manufacturing costs, it achieves a high yield with simple handling for bioreactions.
The bioreactor in accordance with the invention is suitable for different kinds of bioreaction methods including the continuous flow method. It is, however, preferred for methods in which no pumping is used i.e. in which the supply liquid in the supply chamber is not subjected to external pressure during the biochemical reaction. Consequently, the exchange of substances between the chambers occurs solely by diffusion through the membrane, i.e. dialysis. As the concentrations of substances used up during biosynthesis decreases within the central chamber, those substances flow out of the at least one supply chamber in response to the concentration gradient. The same is true in reverse for biosynthesis waste products.
The semi-permeable membrane is preferentially a dialysis membrane. Dialysis membranes have (for the same molecular cutoff) lower mechanical strength than ultrafiltration membranes utilized in the continuous flow method. Nevertheless, the probability of mechanical damage to the membrane of the bioreaction module in accordance with the invention is very low and, in comparison to an ultrafiltration membrane having the same molecular cutoff, a substantially more effective biosynthesis (better yield) is achieved. The molecular cutoff is preferentially at least approximately 10 kD and at most approximately 20 kD.
For some applications, the housing advantageously includes two supply chambers which border a system chamber and are separated therefrom by a first and a second semi-permeable membrane. The housing of such a three chamber module has two supply chamber elements, wherein the first semi-permeable membrane is mounted between the system chamber element and the first supply chamber element and the second semi-permeable membrane is mounted between the system chamber element and the second supply chamber element.
In accordance with an additional preferred embodiment, at least one and preferentially each of the module chambers contain a magnetic stirring element which can be set into rotation by an external magnetic field. The bioreaction module is a disposable item and can be produced ready for use, optionally including the magnetic stirring element.